Ndeye Diop-Bove

Mechanisms of Membrane Transport of Folates into Cells and Across Epithelia

Until recently, the transport of folates into cells and across epithelia has been interpreted primarily within the context of two transporters with high affinity and specificity for folates, the reduced folate carrier and the folate receptors. However, there were discrepancies between the properties of these transporters and characteristics of folate transport in many tissues, most notably the intestinal absorption of folates, in terms of pH dependency and substrate specificity. With the recent cloning of the proton-coupled folate transporter (PCFT) and the demonstration that this transporter is mutated in hereditary folate malabsorption, an autosomal recessive disorder, the molecular basis for this low-pH transport activity is now understood. This review focuses on the properties of PCFT and briefly addresses the two other folate-specific transporters along with other facilitative and ATP-binding cassette (ABC) transporters with folate transport activities. The role of these transporters in the vectorial transport of folates across epithelia is considered.

The Intestinal Absorption of Folates

The properties of intestinal folate absorption were documented decades ago. However, it was only recently that the proton-coupled folate transporter (PCFT) was identified and its critical role in folate transport across the apical brush-border membrane of the proximal small intestine established by the loss-of-function mutations identified in the PCFT gene in subjects with hereditary folate malabsorption and, more recently, by the Pcft-null mouse. This article reviews the current understanding of the properties of PCFT-mediated transport and how they differ from those of the reduced folate carrier. Other processes that contribute to the transport of folates across the enterocyte, along with the contribution of the enterohepatic circulation, are considered. Important unresolved issues are addressed, including the mechanism of intestinal folate absorption in the absence of PCFT and regulation of PCFT gene expression. The impact of a variety of ions, organic molecules, and drugs on PCFT-mediated folate transport is described.

Hypermethylation of the human proton-coupled folate transporter (SLC46A1) minimal trancriptional regulatory region in an antifolate-resistant HeLa cell line

This laboratory recently identified a novel proton-coupled folate transporter (PCFT) that mediates intestinal folate absorption and transport of folates into the central nervous system. The present study focuses on the definition of the minimum transcriptional regulatory region of this gene in HeLa cells and the mechanism(s) underlying the loss of PCFT expression in the methotrexate-resistant HeLa R1-11 cell line. The PCFT transcriptional regulatory controls were localized between −42 and +96 bases from the transcriptional start site using a luciferase-reporter gene system. The promoter is a G + C rich region of 139 nucleotides contained in a CpG island. HeLa R1-11 cells have no mutations in the PCFT open reading frame and its promoter; the transcription/translation machinery is intact because transient transfections in HeLa R1-11 and wild-type HeLa cells produced similar luciferase activities. Hypermethylation at CpG sites within the minimal transcriptional regulatory region was shown in HeLa R1-11 cells as compared with the parental PCFT-competent HeLa cells, using bisulfite conversion and sequence analysis. Treatment with 5-aza-2′-deoxycytidine resulted in a substantial restoration of transport and PCFT mRNA expression and small but significant decreases in methylation in the promoter region. In vitro methylation of the transfected reporter plasmid inhibited luciferase gene expression. Cytogenetics/fluorescence in situ hybridization indicated a loss of half the PCFT gene copies in HeLa R1-11 as compared with PCFT-competent HeLa cells. Taken together, promoter silencing through methylation and gene copy loss accounted for the loss of PCFT activity in antifolate-resistant HeLa R1-11 cells. [Mol Cancer Ther 2009;8(8):2424–31]

Identification of novel mutations in the proton-coupled folate transporter (PCFT-SLC46A1) associated with hereditary folate malabsorption

Hereditary folate malabsorption (HFM) is an autosomal recessive disorder, recently shown to be due to loss-of-function mutations of the proton-coupled folate transporter (PCFT-SLC46A1), resulting in systemic and central nervous system folate deficiency. Data is emerging on the spectrum of PCFT mutations associated with this disorder. In this report, novel mutations are described in three subjects with HFM: A335D/N68Kfs (c.1004C>A/c.204-205delCC), compound heterozygous mutations, and two homozygous PCFT mutations, G338R (c.1012G>C) and E9Gfs (c.17-18insC). Functional assessment of A335D and G338R PCFT mutants transfected into folate transporter-deficient HeLa R1-11 cells indicated a complete loss of transport activity. There were neurological deficiencies in two of the families reported; in particular, late-onset seizures. The importance of early diagnosis and treatment to achieve physiological cerebrospinal fluid folate levels is emphasized.

Properties of the Arg376 residue of the proton-coupled folate transporter (PCFT-SLC46A1) and a glutamine mutant causing hereditary folate malabsorption

The proton-coupled folate transporter (PCFT-SLC46A1) is required for intestinal folate absorption and is mutated in the autosomal recessive disorder, hereditary folate malabsorption (HFM). This report characterizes properties and requirements of the R376 residue in PCFT function, including a R376Q mutant associated with HFM. Gln, Cys, and Ala substitutions resulted in markedly impaired transport of 5-formyltetrahydrofolate (5-FTHF) and 5-methyltetrahydrofolate (5-MTHF) due to an increase in K(m) and decrease in V(max) in HeLa R1-11 transfectants lacking endogenous folate transport function. In contrast, although the influx K(m) for pemetrexed was increased, transport was fully preserved at saturating concentrations and enhanced for the like-charged R376K- and R376H-PCFT. Pemetrexed and 5-FTHF influx mediated by R376Q-PCFT was markedly decreased at pH 5.5 compared with wild-type PCFT. However, while pemetrexed transport was substantially preserved at low pH (4.5-5.0), 5-FTHF transport remained very low. Electrophysiological studies in Xenopus oocytes demonstrated that 1) the R376Q mutant, like wild-type PCFT, transports protons in the absence of folate substrate, and in this respect has channel-like properties; and 2) the influx K(m) mediated by R376Q-PCFT is increased for 5-MTHF, 5-FTHF, and pemetrexed. The data suggest that mutation of the R376 residue to Gln impairs proton binding which, in turn, modulates the folate-binding pocket and depresses the rate of conformational alteration of the carrier, a change that appears to be, in part, substrate dependent.

MUTATION OF THE PROTON-COUPLED FOLATE TRANSPORTER GENE (PCFT-SLC46A1) IN TURKISH SIBLINGS WITH HEREDITARY FOLATE MALABSORPTION

Hereditary folate malabsorption (HFM) is a rare autosomal recessive disorder characterized by systemic and central nervous system folate deficiency. Turkish siblings are reported with the clinical syndrome of HFM, homozygous for deletion of 2 bases (c.204_205 delCC) within the first exon of the proton-coupled folate transporter (PCFT) gene, causing a frameshift. Low blood and cerebrospinal fluid folate levels were detected at ages 3.5 and 1 month. Treatment with parenteral 5-formyltetrahydrofolate resulted in normal development now at ages 3 and 1 year. Extending current knowledge on the phenotypic manifestations of HFM and the PCFT mutation spectrum will provide opportunities to define possible genotype-phenotype correlations and clarify the basis for the phenotypic variability that is characteristic of this disorder.

Random Mutagenesis of the Proton-coupled Folate Transporter (SLC46A1), Clustering of Mutations, and the Bases for Associated Losses of Function

Loss-of-function mutations in the proton-coupled folate transporter (PCFT, SLC46A1) result in the autosomal recessive disorder, hereditary folate malabsorption (HFM). Identification and characterization of HFM mutations provide a wealth of information on the structure-function relationship of this transporter. In the current study, PCR-based random mutagenesis was employed to generate unbiased loss-of-function mutations of PCFT, simulating the spectrum of alterations that might occur in the human disorder. A total of 26 mutations were generated and 4 were identical to HFM mutations. Eleven were base deletion or insertion mutations that led to a frameshift and, along with similar HFM mutations, are predominantly localized to two narrow regions of the pcft gene at the 5′-end. Base substitution mutations identified in the current study and HFM patients were largely distributed across the pcft gene. Elimination of the ATG initiation codon by a one-base substitution (G > A) did not result in a complete lack of translation at the same codon consistent with rare non-ATG translation initiation. Among six missense mutants evaluated, three mutant PCFTs were not detected at the plasma membrane, one mutation resulted in decreased binding to folate substrate, and one had a reduced rate of conformational change associated with substrate translocation. The remaining PCFT mutant had defects in both processes. These results broaden understanding of the regions of the pcft gene prone to base insertion and deletion and inform further approaches to the analysis of the structure-function of PCFT.

Prevalence of a loss-of-function mutation in the proton-coupled folate transporter gene (PCFT-SLC46A1) causing hereditary folate malabsorption in Puerto Rico.

OBJECTIVE To determine whether subjects of Puerto Rican heritage are at increased risk for a specific mutation of the proton-coupled folate transporter (PCFT) causing hereditary folate malabsorption (HFM). STUDY DESIGN Three percent of the births in Puerto Rico in 2005, with additional regional oversampling, were screened for the prevalence of the c.1082G>A; p.Y362_G389 del PCFT gene mutation. Six new subjects of Puerto Rican heritage with the clinical diagnosis of HFM were also assessed for this mutation. RESULTS Six subjects of Puerto Rican heritage with the clinical diagnosis of HFM were all homozygous for the c.1082G>A; p.Y362_G389 del PCFT mutation. Three heterozygote carriers were identified from the 1582 newborn samples randomly selected from births in Puerto Rico in 2005. The carrier frequency for the mutated allele was 0.2% island-wide and 6.3% in Villalba. CONCLUSION These findings are consistent with a common mutation in the PCFT gene causing HFM that has disseminated to Puerto Ricans who have migrated to mainland United States. Because prompt diagnosis and treatment of infants with HFM can prevent the consequences of this disorder, newborn screening should be considered in high-risk populations and physicians should be aware of its prevalence in infants of Puerto Rican ancestry.

Enhanced receptor-mediated endocytosis and cytotoxicity of a folic acid- desacetylvinblastine monohydrazide conjugate in a pemetrexed-resistant cell line lacking folate-specific facilitative carriers but with increased folate receptor expression

The reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptors (FR) are folate-specific transporters. Antifolates currently in the clinic, such as pemetrexed, methotrexate, and pralatrexate, are transported into tumor cells primarily via RFC. Folic acid conjugated to cytotoxics, a new class of antineoplastics, are transported into cells via FR-mediated endocytosis. To better define the role of PCFT in antifolate resistance, a methotrexate-resistant cell line, M160-8, was selected from a HeLa subline in which the RFC gene was deleted and PCFT was highly overexpressed. These cells were cross-resistant to pemetrexed. PCFT function and the PCFT mRNA level in M160-8 cells were barely detectable, and FR-α function and mRNA level were increased as compared with the parent cells. While pemetrexed rapidly associated with FR and was internalized within endosomes in M160-8 cells, consistent with FR-mediated transport, subsequent pemetrexed and (6S)-5-formyltetrahydrofolate export into the cytosol was markedly impaired. In contrast, M160-8 cells were collaterally sensitive to EC0905, a folic acid–desacetylvinblastine monohydrazide conjugate also transported by FR-mediated endocytosis. However, in this case a sulfhydryl bond is cleaved to release the lipophilic cytotoxic moiety into the endosome, which passively diffuses out of the endosome into the cytosol. Hence, resistance to pemetrexed in M160-8 cells was due to entrapment of the drug within the endosome due to the absence of PCFT under conditions in which the FR cycling function was intact.

Biological Role, Properties, and Therapeutic Applications of the Reduced Folate Carrier (RFC-SLC19A1) and the Proton-Coupled Folate Transporter (PCFT-SLC46A1)

The mechanisms by which folates are transported across cell membranes have been an area of research interest for nearly five decades. Major transport systems include the facilitative carriers, the reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT), and the high affinity folate receptors (FRs) α and β which transport folates by endocytosis. RFC is the major transport system in mammalian cells and tissues for folate cofactors and clinically relevant antifolate drugs including methotrexate, raltitrexed, pemetrexed, and pralatrexate. PCFT was identified in 2006 as the mechanism by which folates are transported across the apical brush border of the proximal small intestine. Whereas both PCFT and RFC are widely expressed in tumors, PCFT differs from RFC in its acidic pH optimum which favors transport at the low pH commonly found in the hypoxic microenvironment of solid tumors. Reflecting tumor-specific patterns of expression and/or function, recent studies have focused on the identification of folate-targeted therapeutics with selective transport by PCFT and FRs over RFC. The goal is to circumvent RFC and the potentially toxic consequences of drug transport by RFC in normal tissues. RFC in tumor cells can also influence the pharmacologic activity of PCFT and FR-selective agents by transporting physiological folates which compete for polyglutamylation and binding to intracellular targets. This review focuses on the facilitative pathways of (anti)folate transport, including RFC (SLC19A1) and PCFT (SLC46A1) in relation to their molecular properties, and their physiological and pharmacological roles.